Microbubbles and methods for oxygen delivery

ABSTRACT

Compositions containing a carrier and microbubles encapsulating one or more gases, preferably oxygen, and methods for making and using the compositions are described herein. The microbubbles contain a lipid envelope formed of at least one base lipid and at least one emulsifying agent. The compositions may be administered to a patient to quickly deliver large amounts of oxygen to the patient&#39;s blood supply or directly to a tissue in need of oxygen. The compositions may be administered via injection or as a continuous infusion. The compositions contain a concentrated microbubble suspension, where the suspension contains at least 40 mL oxygen/dL suspension. The microbubbles are preferably less than 20 microns in diameter, more preferably less than 15 microns in diameter. The microbubbles described herein may be administered to a patient in an effective amount to increase in oxygen concentration in the patient&#39;s blood, and/or one or more tissues or organs. The microbubbles may be administered alone or in combination with other treatments as an adjuctive therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 61/073,334, filed Jun.17, 2008; U.S. Ser. No. 61/026,984, filed Feb. 7, 2008; and U.S. Ser.No. 60/975,705, filed Sep. 27, 2007.

The disclosures in the applications listed above are herein incorporatedby reference.

FIELD OF THE INVENTION

This present invention relates to compositions and methods for gasperfusion of tissues, and especially delivery of an effective amount ofoxygen to a patient to alleviate or prevent ischemic injury.

BACKGROUND OF THE INVENTION

Every human cell requires a constant supply of oxygen to maintaincellular structure and homeostasis. This supply is primarily provided byhemoglobin, which carries inspired oxygen from the pulmonary capillariesto the tissues. In cases where a patient's lungs are unable to transferadequate amounts of oxygen to circulating erythrocytes, severe hypoxiaresults and can quickly lead to severe organ injury and death.

Restoration of blood oxygen tension is paramount to resuscitation of themajority of pathophysiologic states. Some clinical states, such as lunginjury, airway obstruction, and intracardiac mixing, exhibit hypoxemiaand desaturation refractory to medical efforts to restore levels ofoxygen saturation sufficient to limit ischemic injury. Ischemic injurymay take place within minutes or seconds of insufficient oxygendelivery. In these conditions, low oxygen tension can result inend-organ dysfunction, failure, and mortality. The ability to augmentoxygenation quickly and non-invasively would have dramatic implicationson the morbidity and mortality from acute hypoxia, in addition to anumber of other clinical situations.

Conventional attempts to restore oxygen levels in patients utilizesupportive therapy of the patient's respiratory system, most commonly byway of mechanical ventilation. However, patients with lung injury,comprising a significant population of intensive care unit patients,have difficulty exchanging oxygen across a damaged alveolar unit. Thisrequires clinicians to increase ventilator pressures, often causingfurther lung injury and systemic inflammation. Significant morbidity andmortality has been associated with ventilator induced lung injury, andbarotrauma to the lungs is often necessitated by inadequate systemicoxygen delivery. The ability to non-invasively supplement even smallpercentages of oxygen delivery may significantly reduce the morbidity ofmechanical ventilation.

Furthermore, emergency efforts to deliver oxygen to a patient are ofteninadequate and/or require too long to take effect, either due to lack ofan adequate airway or overwhelming lung injury. This results inirreversible injury to the brain and other organs. Initiation of rescuetherapy in these patients is burdensome and time consuming, and isavailable only at a limited number of specialized health care centers.There remains a need to quickly deliver oxygen directly to the blood ofpatients, thereby preventing or minimizing irreversible injury due tohypoxemia.

Therefore it is an object of the invention to provide improved methodsfor delivering oxygen to patients, tissues or organs.

It is yet a further object of the invention to provide improvedcompositions for delivering oxygen to patients, tissues or organs.

It is a still further object of the invention to provide improvedmethods for producing compositions for delivering oxygen to patients,tissues or organs.

SUMMARY OF THE INVENTION

Compositions containing a carrier and microbubbles encapsulating one ormore gases, preferably oxygen, and methods for making and using thecompositions are described herein. The microbubbles contain a lipidenvelope formed of at least one base lipid and at least one emulsifyingagent. The compositions may be administered to a patient to quicklydeliver large amounts of oxygen to the patient's blood supply ordirectly to a tissue in need of oxygen.

The compositions may be administered via injection or as a continuousinfusion. The compositions contain a concentrated microbubblesuspension, where the suspension contains at least 40 mL oxygen/dLsuspension. The microbubbles are preferably less than 20 microns indiameter, more preferably less than 15 microns in diameter. Themicrobubbles described herein may be administered to a patient in aneffective amount to increase the oxygen concentration in the patient'sblood, and/or one or more tissues or organs, preferably in an amounteffective to prevent or alleviate ischemic injury. The microbubbles maybe administered alone or in combination with other treatments as anadjuctive therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a microbubble and its lipid envelope.

FIG. 2 is a schematic of a bench-top method for forming microbubbles.

FIGS. 3 A, B, C, and D are graphs showing the mean increase in PO₂ abovethe control PO₂ (mm Hg) as plotted against the ratio of the rate ofunconcentrated microbubble to the rate of blood infusion for each offour microbubble suspensions (C14 (FIG. 3A), C14:16 (FIG. 3B), C16 (FIG.3C), and C18 (FIG. 3D)).

FIG. 4 is a graph of size distribution for the microbubbles, plottingpercent volume versus diameter (μm), as determined by optical sizingfollowing gentle centrifugation (gray line) and following an isolationprocess (black line).

FIG. 5 is a graph of percentage of encapsulated oxygen (% of totalvolume) of microbubbles with four different acyl chain lengths (C14,C15, C16 and C18) following six serial centrifugations at varying speeds(expressed as multiples of gravity force). The data is plotted aspercentage of encapsulated oxygen versus speed (amount x Gravity), withC14 represented by a diamond, C15 represented by a square, C16represented by a triangle and D18 represented By an “x”.

FIG. 6 is a graph of PO₂ (mmHg) versus microbubble infusion rate(mL/min) for infusions of DPPC (C16) oxygenated microbubbles (20 volume% (ml oxygen per dL suspension), mixed in normal saline) in human venousblood.

FIG. 7 is a graph of PCO₂ (mm Hg) (top line) and serum bicarbonateconcentration measurements (mmol/L) (bottom line) versus microbubbleinfusion rate (mL/min) for infusions of DPPC (C16) oxygenatedmicrobubbles (20 volume % (ml oxygen per dL suspension), mixed in normalsaline) in human venous blood.

FIG. 8 is a graph of dissolution time (s) versus radius (μm) for asingle microbubble encapsulating oxygen in a degassed aqueousenvironment.

FIG. 9 is a graph of gas fraction % (mL of oxygen gas per dL ofsuspension) versus time (hours), where time 0 denotes the time ofmicrobubble suspension creation.

DETAILED DESCRIPTION OF THE INVENTION I. Compositions

Compositions containing a carrier and microbubbles encapsulating one ormore gases, preferably where at least one gas is oxygen, foradministration to patients, tissues or organs in need of treatment aredescribed herein. The compositions are particularly preferred forquickly delivering large amounts of oxygen to a patient's blood supplyor directly to a tissue or organ in need of oxygen. The compositions maybe administered via injection or as a continuous infusion.

A. Microbubbles

A typical structure for the microbubbles is illustrated in FIG. 1. Asshown in FIG. 1, the microbubbles contain a gas core surrounded by anenvelope formed from one or more lipids and one or more emulsifyingagents in the form of a lipid monolayer or multilayer. The outer surfaceof the envelope forms a protective film, preferably formed frompolyethylene glycol (see FIG. 1).

1. Envelope

The envelope is in the form of a lipid film, in the form of a monolayeror multilayer, preferably in the form of a monolayer. The lipid film maybe between 1 and 100 nm thick, preferably between 1 and 10 nm thick,most preferably between 2 and 5 nm thick. In one preferred embodiment,the lipid film is a monolayer that is about 10 nm thick. A thin lipidfilm affords a high permeability to oxygen, while preventing a directgas-blood interface.

Preferably the overall charge for the envelope is neutral.

The protective border prevents coalescence of the microbubbles througheither a repulsive electrostatic double layer or a short-range repulsivesteric barrier. The border also decreases recognition and uptake by thereticuloendothelial cells (RES) of the microbubble and inhibitscomplement activation and other immunogenic, toxic or thrombogeniceffects. Typically, the envelope contains from 0.1 to 20% (molar),preferably from 5 to 10% (molar) emulsifying agent. The emulsifyingagent generally contains a hydrophilic portion, typically a hydrophilicpolymer, and a hydrophobic portion. The emulsifying agent or a portionthereof, generally the hydrophilic portion of the emulsifying agent,forms a protective border on the outer surface of the microbubble.Preferably the border is in the form of a brush where the hydrophilicportion of the emulsifying agent extends from the lipid containingportion of the envelope to form a border on the outer surface of themicrobubble.

a. Lipids

A variety of lipids may be used to form the lipid film. The lipids maybe natural or synthetic. Suitable lipids include phospholipids, fattyacids, triacyl glycerols, sphingolipids, terpenes, and waxes. Preferablythe envelope contains one or more phospholipids.

Generally the lipid envelope contains at least one base lipid and atleast one emulsifying agent, where at least a portion of the emulsifyingagent forms a protective film on the outer surface of the envelope.“Base lipid” as used herein refers to the one or more lipids in theenvelope that do not contain a component for forming a protective filmthat reduces surface tension, provides mechanical stability and limitsgas diffusion.

The lipid envelope may contain lipids with acyl chains of varyinglengths and degrees of saturation. The lipid envelope may contain lipidswith a single acyl chain length, or different lipids with different acylchain lengths. In a preferred embodiment, the lipid is a long-chainlipid, preferably a saturated diacyl phosphatidylcholine (Di-C_(n)—PC,where n is between 12 and 24, preferably where n is 16 or 18), whichimparts low surface tension, high stability against envelopedissolution, and low gas permeability prior to administration in vivo.Suitable lipids include phosphocholines, phosphoglycerols, phosphatidicacids, phosphoethanolamines, and phosphoserines. Examples include1,2-Dilauroyl-sn-Glycero-3-Phosphocholine (dilauroylphosphatidylcholine,DLPC), 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine(dimyristoylphosphatidylcholine, DMPC),1,2-Dipentadecanoyl-sn-Glycero-3-Phosphocholine(dipentadecanoylphosphatidylcholine, DPDPC),1,2-dipalmitoyl-sn-Glycero-3-Phosphocholine(dipalmitoylphosphatidylcholine, DPPC),1-Myristoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine(1-myristoyl-2-palmitoylphosphatidylcholine, MPPC),1,2-Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DMPG),1,2-Dimyristoyl-3-Trimethylammonium-Propane, cholesterol and itsderivatives, fatty acids, fatty alcohols, and fatty esters.

i. Acyl Chain Length

Lipids in the envelope may have different acyl chain lengths. The numberof carbons in the acyl chains of the lipids may range from 10 to 24carbons. The average acyl chain length of the lipids in microbubblestypically ranges from 10 to 24 carbons. For example, the average acylchain length of the lipids may be 20, 18, 16, 14, 13, or 12 carbons. Theenvelope may include one or more synthetic lipids with asymmetric acylchains, where one acyl chain is longer than another.

Lipids with longer acyl chain lengths are generally preferred comparedto lipids with shorter chain lengths. Generally lipids with longer chainlengths produce more microbubbles with a greater shelf life. However,the chain length should not be too long, decreasing the rate of gasrelease in vivo.

Generally, longer chained lipids (e.g. 24-carbon vs. 16-carbon) are moreresistant to oxygen passage. Resistance to gas release is also increasedin walls composed of saturated (i.e. no double bonds) versus unsaturatedlipids. Lipids with one or more double bonds contain kinks in the acylchains due to the presence of the double bonds, which createsirregularities in the packing geometry, and thereby allows for gas totransfer out of the microbubble more rapidly.

Increasing or decreasing lipid acyl chain lengths in the microbubble mayresult in changes in the shearing properties of the envelope. Yieldshear and surface viscosity may increase monotonically with hydrophobicchain length. A lipid film comprising lipids having longer chain lengthsmay have decreased permeability to gases compared to one with lipidswith shorter acyl chain lengths. This decreased permeability may beattributed to an increase in attractive dispersion and hydrophobicforces between the hydrophobic tails of adjacent lipid molecules,resulting in a more cohesive lipid film. However, longer acyl chainsgenerally provide greater envelope cohesion, which can improvemechanical strength and reduced gas escape kinetics.

Microbubbles containing lipids with longer acyl chain lengths may bestable in solution and exhibit prolonged persistence when mixed withdesaturated blood.

In some applications for the microbubbles, microbubbles containing shortacyl chain lengths (e.g. ≦C14) may be used for a rapid oxygen releaseprofile. Shorter acyl chain lengths are generally more unstable thanlonger acyl chains. A microbubble with an envelope formed of lipidscontaining short acyl chains may require special mechanical or chemicalprocessing to remain stable in suspension prior to injection into thebloodstream or mixing with another substance. Such special mechanical orchemical processing steps include cooling the suspension and creating ofsuspension just prior to injection.

ii. Phase Transition Temperature

As used herein, the “phase transition temperature” (T_(m)) refers to thetemperature at which lipid assemblies transition from a solid(crystalline) phase to a fluid (liquid crystalline) phase. For example,1,2-Dipentadecanolyl-sn-Glycero-3-Phosphocholine (C15) has a T_(m) of33° C. Thus this lipid is in the solid phase at room temperature andtransitions to the fluid phase when it is injected into the body.

Lipids in the fluid phase exhibit significantly higher thermal motion,creating a higher gas permeability and a significantly higher surfacetension compared to the same lipids in the solid phase. Lipids in solidphase are tightly packed together with minimal lipid motion, making themless permeable to gas transfer. A microbubble containing lipids that arein the fluid phase generally has increased surface tension and gaspermeability, which favors rapid dissolution of the microbubble,compared to the same microbubble containing the same lipids in the solidphase.

Shell cohesion, as a function of acyl chain length, may be representedby the reduced temperature (T_(R)), which is equal to the ratio of theambient temperature (or “working temperature”) (T) to the main phasetransition temperature (T_(m)) of the lipid (in Kelvin). For T_(R)>1,the shell is in an expanded (fluid) state at the working temperature.For T_(R)<1, the shell is in a condensed (solid) state at the workingtemperature. See Table 2 for a list of lipids with their correspondingmain phase transition temperatures (T_(m)) and reduced temperatures(T_(R)).

b. Emulsifying Agent

“Emulsifying agent(s)” refers to the one or more surfactants in theenvelope that contain a molecule aiding lipid adsorption to thegas/liquid interface and stabilizing the microbubble to preventcoalescence. Typically, the surfactant is a hydrophilic polymer attachedto a hydrophobic anchor via one or more covalent bonds. Preferably thehydrophobic anchor is a lipid. The hydrophobic anchor may be an alkylgroup, in the form of a single chain or multiple chains. Typically thealkyl group is 12 to 24 carbons in length. Alternatively, hydrophobicanchors such as sterols, or polymers such as polycaprolactone may beused.

Preferably the hydrophilic polymer in the emulsifying agent ispolyethylene glycol (PEG). Typical weight average molecular weights forPEG range from about 550 Da to 5,000 Da. Alternatively, other moleculescan be in place of PEG. Alternatives include polypropylene glycol,polyvinyl alcohol, poly-N-vinyl pyrrolidone and copolymers thereof,mixed polyalkylene oxides having a solubility of at least one gram/literin aqueous solutions such as some poloxamer nonionic surfactants,neutral water-soluble polysaccharides, including dextran, Ficoll, andderivatized celluloses, non-cationic poly(meth)acrylates, non-cationicpolyacrylates, such as poly(meth)acrylic acid, and esters amide andhydroxyalkyl amides thereof, and combinations thereof.

The envelope may contain a variety of different amounts of base lipidsand emulsifying agents. An optimum ratio of emulsifying agents to baselipids, which lies between a minimum ratio needed to have sufficientamounts of emulsifying agents to aid in lipid adsorption, shield thesurface of the microbubble and prevent coalescence and a maximum ratiowhere lateral repulsion forces due to the presence of the emulsifyingagent begin to significantly disrupt packing of the base lipid, may bedetermined experimentally.

Typically, the envelope contains from 0.1 to 20% (molar), preferablyfrom 5 to 10% (molar) emulsifying agent. Suitable molar ratios ofPEGylated lipids:base lipids include, for example, 1:99, 5:95, 10:90,20:80, and 50:50. Preferably the ratio of PEGylated lipids:base lipidsranges from 5:95 to 10:90.

i. PEGylated Lipids

Emulsifying agents formed of a lipid and PEG are referred to herein as“PEGylated lipids”.

In the preferred embodiment the emulsifying agent is a PEGylated lipid.The PEgylated lipid may contain the same lipid as the base lipid in theenvelope. Alternatively, the PEGylated lipid may contain a lipid that isdifferent from the base lipid in the envelope. Examples of suitablePEGylated lipids include1,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-1000],1,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-2000], and1,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-5000].

3. Optional Molecules

In one embodiment, one or more proteins or polymers may be used in placeof the one or more lipids to stabilize the microbubble.

Optionally, the envelope may include one or more molecules in additionto the lipid(s) and emulsifying agent(s) to stabilize the microbubbles.Suitable stabilizers include polymers and proteins. Suitable polymersinclude lipophilic and amphiphilic polymers. The proteins and polymersmay be within the lipid monolayer or multilayer.

The proteins may be a single protein or a mixture of proteins. Suitableproteins include lipophilic and amphiphilic proteins. Exemplary proteinsinclude lung surfactant proteins, such as SP-A, SP-B, SP-C, or SP-D,synthetic lung surfactant proteins, lung surfactant protein mimetics,and derivatives thereof.

Alternatively the protein may be in a coating on the surface of theenvelope.

2. Gas Core

The gas core contains at least one gas. The gas core does not contain afluorinated gas. The gas must be pharmacologically acceptable, i.e.biocompatible and have minimal toxicity when released. Preferably thegas is able to diffuse through the envelope following administration.Preferably the gas is oxygen.

Other suitable gases include carbon dioxide, nitrogen, nitrous oxide,helium, argon, nitric oxide, xenon, carbon monoxide. These gases may bein the gas core alone or in combination with one or more other gases.For example, the gas core may contain a gas mixture containing oxygenand one or more additional gases.

In another embodiment, the gas contained within the microbubbles may bea biologically useful gas other than oxygen, including, but not limitedto, nitric oxide, and volatile anesthetics, such as isoflurane.

B. Carrier

In one embodiment, the carrier is saline or another physiologicallyacceptable fluid. The carrier should be generally isotonic with blood.Suitable carriers include normal saline, physiological saline or watercontaining one or more dissolved solutes, such as salts or sugars, whichdo not substantially interfere with the formation and/or stability ofthe microbubbles.

In another embodiment, the carrier is a synthetic colloid, such as 6%hetastarch combined with a physiologically balanced crystalloid carrierthat is similar to the plasma electrolyte balance (Hextend®, BioTime,Inc.), or hemoglobin-based oxygen carrier (HBOC), e.g. PolyHeme®(Northfield Laboratories, Evanston, Ill.), Hemopure® (HBOC-201) (BiopureCorp., Cambridge, Mass.), or HemoLink™ (Hb-raffimer) (Hemosol Inc,Toronto, Canada). In this embodiment, after the microbubbles release theoxygen in vivo, they leave behind the lipid envelope, which exhibitsstrong oncotic pressure, and carrier, which serves as a volume expander.

C. Excipients and Other Active Agents

In addition to containing the microbubbles, the carrier may containexcipients or other active agents.

The compositions should be generally isotonic with blood. Thus thecompositions may also contain small amounts of one or more isotonicagents. The isotonic agents are physiological solutions commonly used inmedicine and they comprise aqueous saline solution, e.g. 0.9% NaCl, 2.6%glycerol solution, lactated Ringer's solution, and 5% dextrose solution.The compositions may also be mixed with volume expanders, such asHextend®, hetastarch, albumin, 6% Hydroxyethyl Starch in 0.9% SodiumChloride Infusion (Voluven®), etc. The compositions can also be mixedwith blood (e.g. packed red blood cells) or hemoglobin-based oxygencarriers. Additionally, the compositions can be mixed in a physiologicbuffer (e.g. tris(hydroxymethyl) aminomethane, “THAM”). This isparticularly useful in a clinical situation of impaired ventilation.

Active agents that can be included in the compositions includeantioxidants and other factors known to mitigate injury.

D. Concentration of Microbubbles

The microbubbles are designed to release oxygen in clinicallysignificant amounts quickly following injection, while minimizingbuild-up in the circulation of the components that form the envelope andcarrier.

Thus, the injectable compositions generally contain high concentrationsof microbubbles, in a minimal amount of carrier for the composition tobe injected. Typical concentrations range from 70 to 90% (volumegas/volume injectable composition), preferably 80 to 90%. As shown inthe Examples, suspensions containing from 40 to 70 mL oxygen per dLsuspension have favorable mixing properties.

As shown in the Examples, the volume of the gas core is a function ofthe selection of the length of the acyl chains in the lipids that formthe envelope. Further, the maximum packing density, which is a functionof the microbubble size distribution and the shape and deformability ofthe microbubbles, limits the maximum gas fraction of the suspension.Preferably the volume of the gas core comprises 50% or more of theoverall volume of the suspension. In one preferred embodiment, thevolume of the gas core is 50 to 60% of the overall volume of thesuspension. In another embodiment, lower volume percentages arepreferred. Microbubble suspensions containing less than 50% gas (byvolume), may be useful when resuscitation is desired in trauma, or inmicrovascular flaps being treated with microbubbles.

E. Size

The overall diameter of the microbubbles is selected to provide a highsurface area to volume ratio, thereby favoring rapid transfer of the gasout of the microbubbles. For delivery of oxygen to a patient, typically,the microbubbles have diameters of about 20 microns or smaller,preferably the upper limit for the diameter of the microbubbles rangesfrom 15 microns or smaller, or 10 microns or smaller in order to passthrough the pulmonary capillary bed following intravenous injection.

Preferably, the lipid monolayer is quite thin (e.g. about 10 nm), andthe volume of the gas core comprises 90% or more of the overall volumeof the microbubble, typically comprises between 99.00 to 99.99% of theoverall volume of the microbubble.

F. Stability of Microbubble Suspensions

Microbubbles containing oxygen in their gas core may coarsen and breakdown by ripening (transfer of oxygen from a smaller particle to a largerparticle due to differences in Laplace pressures) or by microbubblecoalescence. The rate at which both of these processes occur isinversely proportional to the envelope cohesiveness, which increaseswith increasing lipid packing density and increasing lipid acyl chainlength. Other factors, such as suspension viscocity, temperature andconcentration of oxygen and other gases in the suspension may alsoaffect stability.

The microbubbles described herein may be designed to be used immediatelyfollowing production. In these embodiments, the microbubbles arerelatively unstable, such as for only a few hours following production.

In other embodiments, the microbubbles are stable in storage for weeksto months at room temperature and standard pressures (e.g. 1 atm) or atlower temperatures, such at refrigeration at 4° C.

II. Methods of Making the Microbubble Compositions

Any suitable method for forming the microbubbles, or precursors for themicrobubbles, may be used. Gas-filled microbubbles form by theadsorption of lipid components in the precursor suspension to the gasliquid interface of entrained gas bodies. This adsorption is generallyaccomplished by high energy conditions, such as amalgamation (intenseshaking) or sonication. Other methods of gas injection may be used toform microbubbles, such as flow focusing, T-junctions orelectrohydrodynamic atomization.

Formation of concentrated microparticle suspensions requires fourgeneral steps: (1) generation of the precursor suspension, (2)dispersion of the gas into the precursor suspension to formmicrobubbles, (3) concentration of the microbubble suspension and (4)size isolation to form a concentrated microbubble suspension withmicrobubbles having diameters below a selected upper size limit.

The microbubble suspensions may be formed on-site, just prior toadministration. Alternatively the microbubble suspensions may be formedand stored for a suitable period of time and then used when needed.

A system for rapidly delivering oxygen to a patient in need thereoftypically includes (a) means for generating a microbubble suspension,and (b) means for administering microbubbles continuously ordiscontinuously to a patient, tissue or organ in need thereof. Means forgenerating microbubble suspensions include sonicator and mechanicalagitators, as described below. The microbubble suspensions can beadministered via injection or by continuous infusion or by any othersuitable means.

A. Methods for Forming Microbubbles

Typical methods of forming microbubbles and microbubble precursors areknown in the art. These methods generally include the first two stepslisted above and may include additional steps.

For example, microbubbles may be formed by mixing the lipids, i.e. baselipid(s) and PEGylated lipids, in a suitable organic solvent, such aschloroform; then evaporating the solvent to form a dry lipid film, andresuspending the lipid film in an aqueous medium and sonicating to formmicrobubbles. (see e.g. U.S. Pat. No. 7,105,151 to Unger er at)

Alternatively, as disclosed in EP 0 077 752 to Schering AG, suspensionsof gas microbubbles can be made by mixing an aqueous solution of asurfactant with a solution of a viscosity enhancer as a stabilizer. Thegas bubbles are then introduced into the mixture by forcing the mixtureof reagents and air through a small aperture. Similarly, suspensions ofgas microbubbles can be formed by dissolving each of the lipids in anaqueous solution, such as sterile phosphate-buffered saline or sterilesaline; then mixing the individual lipid solutions in the desired molarratio to form a precursor solution, next the gas can be added to theprecursor solution by any suitable means, including injecting the gasinto a sealed container containing the precursor solution and agitatingthe solution to form microbubbles. The desired gas or mixture of gases,e.g. oxygen gas, may be perfused through the precursor suspension,thereby oxygenating the precursor solution.

Formation of microparticles is optimal when the suspension is kept cool.

Thus, preferably the precursor suspension is cooled by suitable means.Mechanical agitation has been the main method to create encapsulatedmicrobubbles for biomedical applications, since their inception byFeinstein et al. Feinstein, et al., “Microbubble Dynamics Visualized inthe Intact Capillary Circulation”, J. Amer. College of Card., 4(3):595-600 (1984). Mechanical agitation is a common emulsificationprocedure in which a hydrophobic phase (i.e., gas) is dispersed withinan aqueous surfactant solution by disruption of the interface. Shaking aserum vial with a device similar to a dental amalgamator may be used tofor oxygen microbubbles.

Acoustic emulsification (i.e. sonication) may also be used to agitatethe precursor solution and form microbubbles. Sonication generates largequantities of microbubbles (100 mL×10¹⁰ mL⁻¹) rapidly and reproduciblywithin just a few seconds. In sonication, the sonicator horn istypically placed at the suspension-gas interface. The precursorsuspension is sonicated for a sufficient time period at a sufficientpower to produce the microbubbles. Microbubbles created in this wayfollow a heterogenous size distribution.

The largest microbubbles are the most buoyant and rise to the top of thesuspension, while less buoyant, smaller microbubbles remain motile inthe sonicated suspension. This allows for separation based on differentmigration rates in a gravitational field.

B. Concentration of the Microbubble Suspension and Size Isolation

In a bench scale operation, microparticle suspensions are pumped out viaa port at the bottom of the glass beaker and into a sterile syringe.

To continue production of the microbubbles, another pump simultaneouslyreplaces fresh precursor suspension into the top of the glass beaker.Unprocessed microbubble suspensions created in this way typicallycontain 8-10 mL oxygen per dL of suspension. The amount of oxygen in themicrobubble suspensions can be increased by centrifugation.

A rapid and simple method for concentrating and isolatingsub-populations of lipid coated microbubbles has been developed. Thismethod involves the use of differential centrifugation to isolatesize-selected microbubbles based on their migration in a centrifugalfield.

The relative centrifugal force (RCF) needed for a microbubble size classto rise through the column of length L for a fixed centrifugation timecan be calculated. For example, Stokes' equation for the rise velocityof a buoyant particle relative to the bulk fluid under creeping flowconditions can be used as follows:

$\begin{matrix}{{u_{i} = {\frac{2\left( {\rho_{2} - \rho_{1\; i}} \right)}{9\eta_{2}}r_{i}^{2}g}},} & \left( {{Eq}.\mspace{14mu} 1} \right)\end{matrix}$

where subscript i refers to the microbubble size class, r_(i) is themicrobubble radius and g is the gravitational (centrifugal) accelerationmeasured in RCF. (see Kvale, et alt, “Size fractionation of gas-filledmicrospheres by flotation”, Separations Technology, 6(4):219-226(1996)). The effective viscosity, η₂ *, of the microbubble suspensioncan be calculated using Batchelor and Greene's correlation for themodified fluid viscosity:

$\begin{matrix}{{\frac{\eta_{2}^{*}}{\eta_{2}} = {1 + {2.5\; \Phi} + {7.6\; \Phi^{2}}}},} & \left( {{Eq}.\mspace{14mu} 2} \right) \\{{\Phi = {\sum\limits_{i = 1}^{N_{d}}\; \Phi_{i}}},} & \left( {{Eq}.\mspace{14mu} 3} \right)\end{matrix}$

where Φ is total the microbubble volume fraction for N_(d) size classes.(Batchelo & Green, “Determination of Bulk Stress in a Suspension ofSpherical-Particles to Order C-2”, J. of Fluid Mech., 56: 401-427(1972).) Equations 1-3 can be used to calculate the strength of thecentrifugal field (in RCF) for a given initial size distribution, timeperiod and syringe column length.

Then the centrifuge is run at the rate calculated above to remove thelargest microbubbles, e.g. greater than 20 microns, greater than 15microns, or greater than 10 microns. For example, to remove microbubbleshaving diameters greater than 10 microns, the sample may be run for onecycle at 30 RCF for 1 min. Then the cake is discarded, and theinfranatant which contains the smaller microbubbles is saved.

If smaller microbubbles are desired, the infranatant is redispersed inan appropriate volume of diluent, such as PBS. Then the centrifuge canbe run at a higher speed, as calculated above, to remove the largemicrobubbles from the sample.

Using this method, the amount of gas (e.g. oxygen) in the microbubblesuspensions can be increased 4-fold to 10-fold, or by even greateramounts. For example, an unconcentrated suspension which contained 8-10mL oxygen per dL of suspension, was concentrated to produce amicrobubble suspensions with between 40 and 90 mL per dL of suspension.

1. Size Measurements

The size of the microbubbles can be determined by any suitable device,such as an Accusizer® or Multisizer® III. While the Accusizer® measuressize based on light obscuration and scattering, the Multisizer® utilizeselectrical impedance sensing of the volume of electrolyte displaced bythe microbubble as it passes through an orifice.

Microscopy can be used for direct visual inspection of the microbubblesin the suspension.

Flow cytometry can be used to further characterize the polydispersemicrobubbles. Forward-(FSC) and side-(SSC) light scattering measurementscan be taken. These measurements can also be used to correlate the dataobtained using the Accusizer® or Multisizer® III to better understandthe size distribution of the microbubbles.

III. Kits for Delivering Oxygen

A kit for rapidly delivering oxygen to a patient in need thereof maycontain one or more lipids and one or more emulsifying agent for formingmicrobubbles, a pharmaceutically acceptable carrier, and a source ofoxygen.

These components can be combined to form the microbubbles describedherein. The resulting microbubbles contain a lipid envelope and a gascore. The gas core contains oxygen. The lipid envelope contains one ormore lipids in the form of a lipid film and one or more emulsifyingagents The outer surface of the lipid film forms a protective border.The one or more emulsifying agents contains a group or molecule thatforms the protective border, typically a hydrophilic polymer (e.g. PEG).The suspension of microbubbles contains at least 40% oxygen by volume.

Preferably the kit contains instructions for forming the microbubblesand for administering the microbubbles to a patient, tissue or organ inneed thereof.

The components in the kit are preferably provided in sterile packaging.

IV. Uses for the Microbubbles

The microbubbles may be administered to any patient, tissue or organ inneed of an increase in oxygen concentration in their blood, tissue ororgan. The microbubbles may be administered alone or in combination withother treatments as an adjuctive therapy.

Fully saturated whole blood with physiologic hemoglobin contains 20 mLoxygen per dL. Microparticle suspensions can be manufactured to containbetween 40 and 70 mL oxygen per dL of suspension. Thus, the injection ofone dL of suspension can deliver about 40-70 mL of oxygen directly to atissue or organ in need of immediate oxygenation.

In preferred embodiments, the microbubbles contain oxygen and areadministered to patients experiencing local or systemic hypoxia. Hypoxicor ischemic conditions may arise in a patient as a result of a varietyof mechanisms, including, but not limited to, congenital physical orphysiologic disease or disorders, embolisms, including thromboembolisms,peripheral artery occlusive disease, transient ischemic attacks,strokes, acute trauma, surgical interventions, or exposure to chemicalor environmental agents. The microbubbles are administered in aneffective amount and at suitable rate for increasing or maintaining thePO₂ in a patient following administration. Typically, the microbubblesare administered in an effective amount and at suitable rate to deliveran effective amount of oxygen to a patient to ischemic tissues or todesaturated blood in a time ranging from 0.5 to 30 seconds followingadministration, wherein the amount of oxygen that is delivered iseffective to restore PO₂ levels to normal levels or prevent or alleviateischemic injury. Microbubbles providing immediate release of oxygen areparticularly preferred for acute resuscitations and resuscitation theheart of a patient.

In another embodiment the microbubbles provide sustained release ofoxygen. Such microbubbles may be used to deliver oxygen to the brain andother tissues.

Hemorrhagic Shock and Other Trauma Applications

In acute hemorrhage, resuscitative trauma therapy focuses uponrestoration of circulating blood volume and oxygen carrying capacity. Instates of hypovolemic shock, such as resulting from severe blood loss,the oxygen extraction ratio of peripheral tissues is increased. Theresult is further desaturation of blood returning to the right heart.Models of blunt chest trauma and hemorrhagic shock have suggested thatright ventricular (RV) dysfunction impedes resuscitation efforts.

In late hemorrhagic shock, myocardial ischemia causes impairedcontractility. Volume resuscitation of an ischemic, dysfunctional rightventricle may lead to increased RV end-diastolic volume, causing septalshift into the left ventricle (LV), and decreased LV end-diastolicvolume.

The microbubble suspensions can be injected at an appropriateconcentration and rate to deliver oxygen directly to the myocardium in atime period ranging from 3 to 10 second following injection. Forexample, if the microbubble suspensions contains from 40 to 70 mL oxygenper dL of suspension, the injection of one dL of suspension coulddeliver approximately 40-70 mL of oxygen directly to the myocardium.

Optionally, the microbubble suspension may contain a specializedresuscitation fluid, such as synthetic colloid (e.g. Hextend®) orhemoglobin-based oxygen carrier (HBOC) as the carrier. When themicrobubbles deliver oxygen directly to ischemic tissues, they wouldleave behind their PEG-rich lipid shell (which exhibits favorableoncotic properties) and carrier, serving as a volume expander.

Microbubbles as an Adjunctive therapy

Oxygen-carrying microparticle suspensions are a useful adjunctivetherapy in traumatic injury for several reasons.

In patients with airway failures, injectable microbubbles provide aroute of oxygen administration which allows survival of previouslylethal injuries, such as wounds involving the airways and those causingsevere lung injury. Oxygenated microbubbles provide a highly portableform of intravenous oxygen which may be used to rescue such patients,allowing them to be transported to definitive therapy without ischemicinjuries to other organs.

In patients with cardiac arrest, whether traumatic, ischemic orotherwise, successful resuscitation depends upon establishing a patentairway, ventilating the patient with high oxygen concentrations andadequate chest compressions to provide active pulmonary blood flow. Allof these interventions must be in place in order to raise coronaryarterial oxygen content to maximize the likelihood of return ofspontaneous circulation. Intravenous administration of the microbubblescontaining oxygen may provide a bolus of oxygen to the right side of theheart, in an effective amount to improve right heart function viaendocardial oxygen delivery, improve pulmonary arterial perfusion anddelivery of blood to the left side of the heart compared to nointravenous administration of the microbubbles. Even prior torestoration of appropriate mechanical ventilation, microbubblesadministered via intravenous injection can traverse atelectatic lung andperfuse the left heart. The microbubbles may be delivered to the leftheart in an effective amount to decrease time to return of spontaneouscirculation and improve outcomes in cardiac arrest.

In patients with hemorrhagic shock, in the low cardiac output state, anintravenous injection of rapidly dissolving oxygen microbubbles targetsthe myocardium (the first place the microbubbles reach) and delivers thegas core to surrounding endocardium. As shown in the animal studiesdescribed in the Examples, the microbubbles can deliver an effectiveamount of oxygen to rapidly improve cardiac output compared to notreatment and serve as an adjunct therapy to volume repletion.

A concentrated suspension of microbubbles may be added directly toexisting volume expanders in the field, which may improve cardiovascularand cerebral resuscitation. This may serve as a useful adjunctivetherapy to rescue patients with severe hemorrhagic shock, whenmyocardial ischemia leads to dysfunction, inadequate pulmonary bloodflow and systemic desaturation. The microbubble suspensions can bedesign to be stable for extended periods of time, ranging from days toweeks.

Microvascular Operations

Microbubbles having diameters of less than 5 microns in a diluent suchas Dextran may be administered via direct intraarterial injection.Microbubbles that are infused towards at-risk tissues or a recentmicrovascular operation, may deliver an effective amount of oxygen toimprove rheology and oxygen content of the perfusate, and therebyimprove oxygen delivery in these settings.

As a corollary, in circumstances of focal low flow states, such asnear-amputations, microbubbles with prolonged bloodstream persistence,such as from about 15 to about 45 seconds following administration, maybe designed such that they circulate in vivo until local conditionsfavor diffusion of oxygen from the microbubble core into ischemictissues (i.e. when they are surrounded by a hypoxemic milieu). Becausethe oxygen content of microparticle suspensions upon injection isgreater than that of saturated whole blood (40-60 mL O₂/dL suspensionvs. 20 mL O₂/dL blood), continuous microbubble infusions may be used toraise the oxygen content of circulating blood.

Carbon Monoxide Poisoning

Oxygenated microbubble suspensions carry within them high concentrationsof oxygen. As such, they may be effective in displacing hemoglobinscavengers, such as carbon monoxide. In cases of severe carbon monoxidepoisoning, circulating microbubbles could deliver oxygen directly to thetissues in an effective amount to improve hemoglobin function, providinga portable temporizing therapy for patients with impaired hemoglobinfunction.

Traumatic Brain Injury

Infusion of oxygen-bearing microbubbles into the cerebral circulationmay decrease neuronal death at the ischemic penumbra. Given the improvedoxygen content of microbubble suspensions over that of whole blood,patients with impaired cerebral blood flow, e.g. in traumatic braininjury or intracranial hypertension, directed administration ofoxygenated microbubbles into a carotid artery would increase the oxygencontent (CaO₂) of blood flow directed to the brain, and may balance thedecrease in flow with an improvement in oxygen content. Microbubbles maybe mixed in a buffered, low viscocity solution (e.g. THAM) and maycontain antioxidants and other factors known to mitigate neuronal injury(e.g. DHA). The polyethylene glycol moiety of the lipids utilized in themicrobubble envelope exerts significant oncotic pressure, which maydecrease vasogenic edema.

Cyanotic Congenital Heart Disease

A unique feature of congenital heart disease is partial or completemixing of saturated and desaturated blood. In perioperative states,systemic desaturation can lead to significant cerebral and myocardialdysfunction. For example, frequently patients with hypoplastic leftheart syndrome require extracorporeal life support in the perioperativeperiod primarily to prevent death due to hypoxemia and the concomitantmyocardial dysfunction. ELSO. Extracorporeal Life Support RegistryReport, International Summary; 2008 January, 2008.

Microbubbles containing oxygen may be administered intravenously in aneffective amount to raise mixed venous oxygen content, systemic oxygencontent, and improve myocardial function in patients in a perioperativestates. Thus the microbubbles can be administered in place of a moreinvasive use of extracorporeal life support device.

Pulmonary Hypertension

Pulmonary hypertension remains a health care problem with few effectivetherapies. Oxygen is known to be a potent pulmonary vasodilator. Thus,the microbubbles containing oxygen may be administered intravenously inan effective amount to raise the oxygen content in pre-capillarypulmonary arterioles and improve pulmonary vasodilation compared to notreatment in patients with pulmonary hypertension.

Acute Respiratory Distress Syndrome (ARDS)

Refractory hypoxemia is the hallmark of acute lung injury and ARDS.Profound hypoxemia accounts for 10% of the mortality of this commondisorder. Meade et al, “Ventilation strategy using low tidal volumes,recruitment maneuvers, and high positive end-expiratory pressure foracute lung injury and acute respiratory distress syndrome: a randomizedcontrolled trial.” JAMA, 299(6):637-45 (2008). Microbubbles containingoxygen may be administered intravenously in an effective amount toalleviate the hypoxemia associated with severe intrapulmonary shuntingand decrease the mortality and morbidity of ARDS.

Delivery of Microbubbles to Fetuses, Neonates and Infants

The microbubbles may be administered to a fetus, neonate, or infant inneed of additional oxygen. The microbubbles may be administered to lowbirth weight infants or premature infants. In one embodiment, themicrobubbles are administered in an effective amount to ensure that thefetus, neonate, or infant is receiving sufficient oxygen, particularlyto ensure that the brain of the fetus, neonate or infant receivessufficient oxygen for development and maintenance of normal function.

If a mother is experiencing preeclampsia, the baby must be born.Optionally, the microbubbles can be administered to the baby, mother, orboth in effective amount to deliver an effective amount of oxygen tomaintain normal normal bodily functions when the mother is experiencingpreeclampsia.

Neonates with hypoxic ischemic brain injury at the time of birth oftensuffer from extensive brain injury, manifested as cerebral palsy. Thismay occur due to even brief periods of hypoxia during the peripartumperiod. In clinical situations where this is appreciated prior todelivery, such as a nuchal cord or placental abruption, injection ofmicrobubbles into the unbilical circulation or into the dural space mayavert critical hypoxia and may ameliorate some forms of hypoxic ischemicbrain injury in this setting.

Delivery of Other Medical Gases

In another embodiment, the gas contained within the microbubbles may bea biologically useful gas other than oxygen, including, but not limitedto, nitric oxide, and volatile anesthetics, such as isoflorane.

Volatile anesthetics may be included in the gas core in the microbubblesdescribed herein in place of, or in addition to, oxygen. In thisembodiment, the microbubbles may be administered in an effective amountto serve as a sedative, antiepileptic drug or bronchodilator. Forexample, microbubbles containing isoflorane in place of oxygen can bedelivered to diseased airways from the pulmonary capillary to the distalbronchioles. Administration of microbubbles containing isoflorane may beused as an adjunctive therapy for treatment of patients with severeasthma.

Microbubbles containing nitric oxide, a pulmonary vasodilator, in thegas core in place of oxygen may be administered to patients in aneffective amount to deliver this pulmonary vasodilator to pulmonaryarterioles and alleviate pulmonary vasodilation.

Finally, microbubbles containing antiepileptic drugs in the gas core inplace of oxygen can be manufactured, such as by using the methodsdescribed herein, to have sizes suitable for crossing the blood-brainbarrier. The microbubbles can be used to deliver doses of drug that arelower than the standard systemic dose of the antiepileptic drug yetachieve the same effect. Microbubbles containing antiepileptic drugs inthe core may administered to patients in need of treatment in aneffective amount to improve delivery of these drugs to epileptogenicareas and minimize adverse effects associated with systemicadministration of higher doses of the drug.

B. Methods of Administration

The compositions containing microbubble suspensions may be administeredlocally or systemically, depending on the condition to be treated. Thecompositions are typically administered via injection. In someembodiments the compositions can be administered as continuousinfusions. In some embodiments the compositions are administeredintravenously or intraarterially. In others, the compositions areadministered directly to the tissue or organ in need of treatment.

In one embodiment, the microbubble suspensions are stable in storage forprolonged periods of time, and may be withdrawn and directly injectedwithout further alterations of the solution.

In another embodiment, the microbubbles may be formed just prior toadministration, e.g. within seconds or minutes of injection, by asuitable device. The methods disclosed herein allow for rapid productionof oxygen-containing microbubbles for use in clinical settings or in thefield.

C. Rates of Administration

The volume of the gas-filled microbubble suspension to be administeredis a function of a number of factors including, the method ofadministration, the gas percentage of the microbubble suspension, andthe age, sex, weight, oxygen or carbon dioxide tension, blood pressure,systemic venous return, pulmonary vascular resistance, and physicalcondition of the patient to be treated.

The whole body oxygen consumption of an adult at rest is approximately200 mL oxygen per minute. Thus, in the setting of an acute airwayobstruction, for example, infusion of 200 mL/minute of oxygen wouldprevent critical ischemic injury. For example microbubble suspensionscontaining 70 mL/dL of suspension can be administered at 285 mL/minuteto transfer 200 mL/minute of oxygen in vivo. Since most of thesuspension contains oxygen gas, most of the volume decreases followingadministration and release of the gas. Values of co-administered volumesfor physiologically relevant oxygen demands are shown below in Table 1.Additionally, when used in the setting of an acute resuscitation or inorgan-targeted oxygen delivery, volumes of co-infusate may be muchlower, For example, a 10 mL bolus of 50% (volume gas/volume suspension)microbubbles in adults may provide a suitable amount of oxygen toimprove the survival of the organ.

TABLE 1 List of Administration Rates for Carrier and Lipids toAdminister 100 mL/min or 200 mL/min of Oxygen O₂ Delivery 70 Vol % 90Vol % Carrier Volume 100 mL/min 30 mL/min 10 mL/min 200 mL/min 60 mL/min20 mL/min Lipid Volume 100 mL/min 0.054 mL/min 0.054 mL/min 200 mL/min0.108 mL/min 0.108 mL/min

D. Gas Release

The microbubbles are preferably designed to release the gas encapsulatedtherein quickly following administration in vivo. Typical release timesrange from 0.5 seconds to 1 minute, with shorter time periods, such asfrom 0.5 to 30 seconds, more preferably from 0.5 to 10 seconds, beingpreferred for acute resuscitations and resuscitations of the heart andwith longer time periods being preferred for delivery of oxygen to thebrain.

In some embodiments, the microbubbles are designed to persist in vivountil they reach hypoxic tissue, at which time they will release theencapsulated oxygen and the lipid envelope with collapse.

As oxygen is released and the encapsulating envelope collapses, thelipid material sheds as micelles and vesicles, typically having sizesranging from 10 nm to 100 nm, which then undergo hepatic metabolism. Dueto the small size of the lipid film relative to the radius of themicrobubble, the effective volume of lipid is approximately onethousandth of one percent of the volume of suspension.

The lipid material does not persist in vivo for a sufficient time tocarry carbon dioxide or other gases to the lungs. The microbubblesgenerally release the encapsulated gas and the gas is absorbed byhemoglobin prior to the first circulation into the pulmonaryvasculature. In a healthy adult patient with a normal cardiac output,the release of the encapsulated gas typically occurs from 4 to 5 secondsfollowing injection, or faster.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLES Example 1 Formation of Concentrated Microbubble Suspensions

Concentrated microparticle suspensions were formed using the followingfour main steps: (1) generation of the precursor suspension, (2)sonication, (3) concentration and (4) size isolation.

1. Generation of precursor suspension. Microparticle suspensions usingeach of the lipids listed in Table 2 below were created. The base lipidswere received in powder form (Avanti Polar Lipids, Alabaster, Ala.) anddissolved in sterile saline to create a stock solution. The same wasdone with PEGylated lipid. Base lipids were mixed with the PEGylatedlipid in a 95-5 molar ratio. The final concentration of each precursorsolution was 3 mg total lipid/mL. Lipid precursor solutions were storedat 4° C.

TABLE 2 List of Base Lipids and PEGylated Lipids with TransitionTemperature (T_(m)) and Reduced Temperature (T_(R)) T_(m) T_(R) at T_(R)at Microbubble Lipid Components Abbreviation (° C.) 25° C. 37° C. BaseLipids 1,2-Dilauroyl-sn-Glycero-3- C12 −1 1.10 1.14 Phosphocholine1,2-Dimyristoyl-sn-Glycero-3- C14 23 1.01 1.05 Phosphocholine1,2-Dipentadecanoyl-sn- C15 33 0.97 1.01 Glycero-3-Phosphocholine1,2-Dipalmitoyl-sn-Glycero-3- C16 41 0.95 0.99 Phosphocholine1,2-Distearoyl-sn-Glycero-3- C18 55 0.91 0.95 Phosphoethanolamine1,2-diarachidoyl-sn-glycero-3- C20 phosphocholine1,2-didocosanoyl-sn-glycero-3- C22 phosphocholine PEGylated Lipid1,2-Dimyristoyl-sn-Glycero-3- 14:0PE-PEG N/A N/A N/APhosphoethanolamine-N- [Methoxy(Polyethylene glycol)- 550]

2. Sonication. Gas-filled microbubbles self-assemble to form lipidmonolayers when lipid precursor suspensions are mixed with a gas underhigh energy conditions.

A schematic of the instrumentation used for the sonication step isillustrated in FIG. 2.

As shown in FIG. 2, precursor suspensions were stored in a large glasscontainer (10) through which oxygen gas was perfused, oxygenating theprecursor solution. This solution was then moved via roller pump (12)into a beaker (14) surrounded by a cold water bath (Radnoti GlassTechnology, Monrovia, Calif.) (16), which counters the heat created bythe sonicator. Formation of microparticles is optimal when thesuspension is kept cool. The sonicator (Branson Sonifier 250A, BransonUltrasonics, Danbury, Conn.) was operated continuously at maximal power.The ⅝″ sonicator horn (18) was placed at the suspension-gas interface.Pure oxygen gas was flowed continuously over the suspension at 6 litersper minute, creating a pure oxygen hood. The sonicator was maintainedwithin a sound enclosure.

Microbubbles formed in this way follow a heterogenous size distribution.The largest microbubbles are the most buoyant (macrobubbles), which riseto the top of the suspension. Smaller microbbles are less buoyant andremain motile in the sonicated suspension. Microbubble suspensions werepumped out via a port (20) at the bottom of the glass beaker and into asterile syringe (22). Another roller pump (not shown in FIG. 2)simultaneously replaced fresh precursor suspension into the top (24) ofthe glass beaker (10). This method allowed for the formation of 300mL/minute of fresh microbubble suspensions.

The oxygen content of the microbubble suspension was measured asfollows. A syringe was filled with a known volume of microparticles. Thedifference in weight of the filled syringe from that of the emptysyringe was divided by the density of the precursor suspension,estimating the volume of fluid in the syringe. The remainder of thevolume may be assumed to be gas, which is weightless. This method isreferred to herein as the “Oxygen Content Determination Method”.Unprocessed microbubble suspensions created in this way contained 10-20mL oxygen per dL of suspension.

3. Centrifugation. Unprocessed microbubble suspensions were gathered ina batch of 60 mL syringes (Beckton-Dickenson) which were modified byshortening the plungers, so that the syringes could fit in a centrifuge(Beckman table top centrifuge). Syringes were directly filled viapressure from the pump (26) (see FIG. 2), and were sealed with a capfollowing removal from the system described above. Each batch of 16syringes were then placed in the centrifuge for 4 minutes at 500×G. Thisforce separated macrobubbles (least dependent), with diameters greaterthan about 20 to 30 microns, from smaller microbubbles (intermediate),with diameters up to about 20 microns, from infranatant lipid solution(most dependent). After infranatant portions were recycled into theprecursor solution, the thin, heterogenous microbubble layers inside ofeach syringe were combined into one 60 mL syringe using a T connector.When sized optically, these particles were heterogenous in size, rangingfrom 5-30 microns in diameter (see FIG. 4, gray line).

The ability to withstand centrifugation and create a concentratedmicroparticle suspension is a function of microbubble acyl chain length(see FIG. 5). As shown in FIG. 5, the microbubbles formed with lipidswith longer acyl chain lengths (e.g.1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine and1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine) encapsulated greatervolumes of oxygen than those formed from lipids with shorter acyl chains(e.g. 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine and1,2-Dipentadecanoyl-sn-Glycero-3-Phosphocholine). The microbubblesformed with C16 and C18 lipids as the base lipids contained 50-60%oxygen in the concentrated suspension (see lines containing triangles(C16) and “x” (C18) in FIG. 4. In contrast, the microbubbles formed withC14 and C15 lipids as the base lipids contained only 20-20% oxygen inthe concentrated suspension (see lines containing circles (C14) andsquares (C15) in FIG. 5). The oxygen content of the microbubblesuspensions was determined using the Oxygen Content Determination Methoddescribed above. It is expected that microbubbles formed of lipids withlonger acyl chains will obtain the same amounts or even greater amountsof oxygen following the concentration step.

4. Size Isolation. To isolate the desired particle sizes, 40 mLconcentrated particles were resuspended in 20 mL of oxygenated saline ina 60 mL syringe. The syringe was left in an inverted position for 10minutes at room temperature. Microbubbles layer themselves according tothe Stokes approximation for the velocity reduction of a moving spheredue to viscous drag in creeping flow, Using this technique, particles ofless than 15 microns were isolated by capturing only the suspension inthe inferior 6.8 cm of the syringe (total length of each syringe was 7.2cm).

Following removal of the larger microbubbles, the remaining microbubbleswere reconcentrated using a final, low speed centrifugation process(200×G). Results of a 15 micron isolation (following reconcentration)are shown in FIG. 4, black line.

This method was used to produce a concentrated microbubble suspension ata concentration of 80 mL oxygen per dL suspension by weight, asdetermined using the Oxygen Content Determination Method describedabove.

Example 2 Efficiency and Rate of Gas Transfer to Desaturated HumanHemoglobin

The base lipids used in the preparation of the microbubbles were1,2-Dilauroyl-sn-Glycero-3-Phosphocholine;1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine;1,2-Dipentadecanoyl-sn-Glycero-3-Phosphocholine;1,2-dipalmitoyl-sn-Glycero-3-Phosphocholine;1-Miristoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine;1,2-Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)]; and1,2-Dimyristoyl-3-Trimethylammonium-Propane. The PEGylated lipid used inthe microbubbles was1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-5000]. All lipids were purchased from Avanti Polar Lipids,Alabaster, Ala.

Venous whole blood was withdrawn from healthy donors and stored inheparinized syringes. The mean PO₂ of the venous blood was 52.1 mm Hg.

Formation of the Microbubble Suspensions

Each of the above listed lipids was dissolved in sterilephosphate-buffered saline via sonication at high power for 10 minutes,yielding a stable solution of dissolved, concentrated lipid. Base lipidswere mixed with the PEGylated lipid in a 95:5 molar ratio to form aprecursor solution. Each precursor solution was placed in a sealedbeaker, the head space of which was made 100% oxygen by oxygen washout.A sonicator (Branson Sonifier Model 150, Danbury, Conn.) was used tosonicate each precursor solution for 5 seconds, yielding a richoxygen-filled microbubble suspension. The microbubbles were withdrawnfrom the dependent portion of the beaker into a 60 mL syringe, avoidingthe more buoyant macrobubbles which floated to the top. Alternatively,the microbubbles were withdrawn from the beaker via a roller pump, theaffluent line of which was placed in the inferior-most position withinthe beaker.

Microbubble suspensions were infused at incrementally increasing rates;0.2, 0.4, 0.6, 0.8 and 1 mL/min, while desaturated erythrocytres areinfused at 5 mL/min. Infusions were run for two minutes at each rate,and the blood was collected in triplicate at each rate during in thefinal minute for analysis. Blood and microbubbles were mixed byconvection in a circuit composed of gas-impermeable tubing (Tygon,FEP-lined tubing, Cole Parmer, Vernon Hills, Ill.).

PO₂ of the blood was measured following at 1.5 mL of tubing, whichrepresented approximately 3.8 seconds of mixing time. PO₂ wasimmediately measured by conventional blood gas techniques.

In FIGS. 3A, B, C, and D, the mean increase in PO₂ above the control PO₂(mm Hg) is plotted against the ratio of the rate of unconcentratednanoparticle to the rate of blood infusion for each of four nanoparticlesuspensions tested. As shown in FIG. 3, C14 (p=0.01), C15 (p=0.07) andC16 (p=0.004) raised PO₂ above control rates to a statistically andclinically significant extent. C14-based particles increased PO₂ by 50mmHg to greater than 500 mm Hg (depending upon infusion rate). C14:16was ineffective in transferring oxygen. C18 displayed macroscopicevidence of nanoparticle persistence by way of visible nanoparticlesuspension in the tubing (which is white and starkly contrasts blood) aswell as microscopically.

Example 3 Comparison of Microbubbles Encapsulating Oxygen andUnencapsulated Oxygen in Desaturated, Venous Whole Blood Tested In Vitro

The effect of oxygen-bearing microbubbles containing DPPC as the baselipid and PEG stearate as the emulsifying agent in a molar ration of95:5 (base lipid: emulsifying agent), which were prepared according tothe method of Example 1 without the concentration and size isolationsteps, on a 2 mL sample of desaturated, venous whole blood from ahealthy human volunteer was studied. In one test tube, 0.2 mL of themicrobubbles were added to 2 mL of desaturated, venous whole blood. To asecond test tube, 0.2 mL of pure oxygen gas (unencapsulated) was addedto 2 mL of desaturated, venous whole blood.

The test tubes were inverted once, and co-oximetry and blood gas valuesmeasurements were taken using a Radiometer ABL800 X blood gas machine.

The test tube containing microbubbles had a pH of 7.31, PCO₂ of 50 mmHg,PO₂ of 248 mmHG, and the amount of oxygenated hemoglobin (oxyHb) wasmeasured at 99.8%. In contrast, the test tube containing unencapsulatedoxygen had a pH of 7.33, PCO₂ of 59 mmHg, PO₂ of 33 mmHG, and the amountof oxygenated hemoglobin (oxyHb) was measured at 59%.

Thus oxygen was effectively and rapidly transferred from the gas core ofthe microbubbles to hemoglobin following a single inversion of the testtube. While much less oxygen was transferred into the blood byadministering oxygen gas along. Light microscopy showed no evidence ofgross hemolysis.

Example 4 Oxygen Release Kinetics Studies (In Vitro)

Microbubble Formation

For this study, microbubbles containing DPPC as the base lipid and PEGstearate as the emulsifying agent in a molar ration of 95:5 (base lipid:emulsifying agent), which were prepared according to the method ofExample 1 without the concentration and size isolation steps. Followingsonication, the microbubbles were removed by a pump and co-infused,without further processing or concentration steps, with venous humanblood at varying rates. The microbubbles produced in this manner had aconcentration of only 2-5 mL of oxygen per 50 mL of suspension.

The following rates were tested: 1 ml suspension/min, 2 mlsuspension/min, 4 ml suspension/min and 5 ml suspension/min.

Analysis Methods

The oxygen release kinetics of the microbubbles were tested for adose-response relationship. Desaturated, whole blood was withdrawn fromhealthy volunteers and co-infused with the oxygenated microbubbles intoa glass circuit designed to emulate convective blood flow. Temperaturewas maintained at 36° C. Blood was infused at 10 mL/minute.

Oxygen tension was measured five times at each rate, and the increase inPO₂ above baseline was plotted against infusion rate. Blood pH was alsomeasured. PCO₂ (mm Hg) and serum bicarbonate concentrations (mmol/L) forthe blood samples were also measured.

There was no macro- or microscopic evidence of frothing, clotting orhemolysis. There was no evidence of microbubbles by optical sizing orlight microscopy in post-infusion samples.

Results

FIG. 6 shows results of the PO₂ measurements with DPPC (C16) and PEGstearate oxygenated microbubbles (20 volume % (ml oxygen per dLsuspension), mixed in normal saline). FIG. 7 shows the results of thePCO₂ (mm Hg) and serum bicarbonate concentration (mmol.L) measurementsfor the microbubbles.

Microbubbles formed with C16 as the base lipid increased PO₂ by 50 mm Hgto greater than 500 mm Hg (depending upon infusion rate). As shown inFIG. 7, serum bicarbonate levels decreased with decreasing PCO₂measurements, which decreased as the microbubble infusion rateincreased.

Blood pH remained stable over time for each infusion rate. Blood pHranges from about 7.25 to 7.30 for all of the experiments. This is dueto the buffering capabilities of serum bicarbonate, which was noted todecrease with decreasing amounts of carbon dioxide (see FIG. 7).

This may be due to the unique behavior of hemoglobin as described by thehemoglobin dissociation curve. In the presence of high oxygen tension,hemoglobin preferentially sheds carbon dioxide and binds to oxygen.‘Shed’ carbon dioxide dissolves into plasma (therefore, is nothemoglobin-bound and PCO₂ decreases) and is converted to carbonic acid(in turn buffered by bicarbonate, which decreases with increasingdissolved CO₂). Within the physiologic limits allowed by serumbicarbonate and other buffers, this allows for transfer of oxygen toerythrocytes in the bloodstream and the release of carbon dioxidewithout the development of acidosis

Example 5 Effect of Microbubble Size on Dissolution Time (In Vitro)

The effect of microbubble radius as an independent predictor ofmicroparticle lifetime in a degassed aqueous environment was estimatedby numerical simulation as follows. The partial pressures of the gasesin the aqueous environment were held constant at P_(O2) of 18 mmHg,P_(CO2) of 45 mmHg, and P_(N2) of 592.8 mmHg.

To model the gas release kinetics, a mass balance was performed aboutthe microbubble as gases were either entering or leaving the microbubblefrom the surroundings, depending on the difference in partial pressuresbetween the interior and exterior phases. Gas flux was positive in thedirection of decreasing partial pressure. The microbubble was initiallypure oxygen. Diffusion in the liquid phase was modeled to take placeover a thin film equal to the microbubble radius. This is equivalent tothe Sherwood number for a purely dissolving sphere. At the surface, themicrobubble partial pressure was given by Henry's Law using the pressureinside the microbubble, which was the sum of the hydrostatic and Laplacepressures. At the diffusion layer boundary, the partial pressure wasassumed to be equal to the bulk phase partial pressure. Diffusion wasmodeled according to Fick's Law. A component balance was taken at eachtime step for each gas species and summed to provide the total pressureand volume of gas inside the microbubble. The dynamics of microbubbledissolution were thus determined by numerical simulation. Thedissolution time was given by the time to reach a radius of zero.

Results

Dissolution time was estimated to increase exponentially with increasingmicrobubble radius (FIG. 8). For microbubbles with a radius of about 2μm, the gas was determined to be released and the bubble was determinedto dissolve in less than 2 seconds in the aqueous environment. While formicrobubbles with a radius of about 150 mm, it was determined to takeabout 5,000 seconds for the microbubble to dissolve. Based on theseestimates, the preferred microbubble diameter appears to be 10-12microns or less. This size range is estimated to provide a dissolutiontime of about 10 seconds or less.

Example 6 Particle Stability Testing

In some applications, it is helpful to provide stable microbubbles thatcan be stored for weeks or months following production. This study wasdirected at determining the longevity of stable microbubble suspensions.

60 mL syringes (Becton-Dickenson) were filled with 80 mL oxygen/dLmicrobubble suspension. The microbubble suspensions were formed asdescribed in Example 1 and contained 95:5 molar C16 to 14:0βE-PEG.Syringes were capped with a standard air-tight cap, and were stored atroom temperature or 4° C., as noted.

Gas fraction was measured by the change in weight of the syringe dividedby the volume of suspension within it. Prior to each measurement, largemicrobubbles were removed by mechanically agitating the sample, bringingthe larger (more buoyant) microbubbles to the top of the syringe,allowing for expulsion from the syringe.

Particle sizing at each of the measurement points revealed no changes inparticle size distribution following removal of buoyant microbubbles.

As shown in FIG. 9, microbubbles composed of DPPC (C16) as the baselipid can be stored at room temperature and are preserved at 80 mLoxygen per dL suspension for at least 8 hours.

Microbubbles composed of C18 exhibit significant volume loss using thecreation and storage techniques used in this study.

Example 7 In Vivo Tests

The following experimental results provide data from two in vivoexperiments. To test the ability of microparticles to diffuse oxygeninto ischemic tissues and desaturated blood, test were conducted in arabbit model of hypoxemic ventilation. All animal experiments wereapproved by the Institutional Animal Care and Use Committee ofChildren's Hospital Boston. Animals were housed and surgical procedurestook place under the supervision of a staff veterinarian in the AnimalResearch facility of Children's Hospital Boston (ARCH).

Animal Preparation: Two male New Zealand rabbits (3-4 kg) were purchased(Milbrook Farms, Milbrook, Mass.) and were allowed a three dayacclimation period. Animals received appropriate intravenous analgesiaand sedation, endotracheal intubation, and surgical placement of centralvenous catheters (PE50 catheters) into the right internal jugular vein,femoral vein and femoral artery. Animals were allowed to breathe roomair spontaneously until the start of the experimental procedure.Sedation and analgesia were maintained by continuous infusions ofFentanyl and Midazolam.

Microbubble Preparation: The microbubbles utilized for this study werecomposed of 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (C16) mixed in a95:5 molar ratio with 14:0 PE-PEG (550), as described in Example 1. 200mL of microbubbles were formed within 1 hour. The resultant suspensionexhibited a concentration of 80 mL oxygen/dL suspension, and particlesizing via microscopy revealed that the microbubbles had diameters ofless than 15 microns.

Experimental Procedure: Animals were ventilated using 15% oxygen, 85%nitrogen. Animals were provided a mandatory respiratory rate to ensureadequate minute ventilation. End-tidal carbon dioxide and end-tidaloxygen content, pulse oximetry, blood pressure and EKG were monitoredcontinuously. In the animals tested (n=2), the animals became pulselessjust following the induction of acute hypoxemia. The thorax andpericardium were opened to observe the heart under direct visualization.Ventilation was discontinued to maximize visualization. Microbubblesuspensions were then injected via internal jugular catheter in 1 mLaliquots to provide rescue oxygenation.

Experimental Results Upon opening the thorax, rabbit hearts were foundto be cyanotic, dyskinetic and severely bradycardic (heart rate of about10 beats/minute), following loss of pulsatility. Microbubble suspensionswere then injected into the internal jugular catheter, and were seenentering the right atrium (microbubble suspensions are white and wereeasily visualized in the bloodstream). There was no other drug mixedwith the microbubble suspension.

Immediately upon entering the atrium, atrial contractions increasedsignificantly in force and frequency. Seconds later, right ventricularfunction and rate improved heart rate of about 100 beats/minute).Microbubbles were subsequently noted in the left atrium, with subsequentimproved left atrial and ventricular function after about 2 minutesfollowing injection.

In both animals, the heart was perfused with aliquots of microbubblesuspensions. The lungs remained collapsed throughout the duration of theexperiment.

In the first animal, ventricular function was maintained for 90 minutesin this manner.

In the second animal, in the process of exposing the thorax, thesuperior vena cava was unintentionally lacerated. A hemorrhage ofapproximately 20 mL/kg in the setting of severe bradycardia occurred.The microbubble infusion was injected into the internal jugular venouscatheter. As in the first animal experiment, microbubbles were alsovisualized entering the right atrium. Atrial and ventricular functionwere noted to improve in chronotropy and inotropy following microbubblecontact with the myocardium.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for delivering oxygen to a patient, tissue or organ,comprising administering to the patient, tissue or organ a composition,comprising a suspension comprising microbubbles and a carrier, whereinthe microbubbles comprise a lipid envelope and a gas core, wherein thegas core comprises oxygen, wherein the lipid envelope comprises one ormore lipids in the form of a lipid film, and one or more emulsifyingagents, wherein the outer surface of the lipid film forms a protectiveborder, wherein the one or more emulsifying agents comprises a group ormolecule that forms the protective border, and wherein the suspensioncomprises at least 40% oxygen by volume, wherein the composition isadministered in an effective amount to increase the concentration ofoxygen in the patient's blood, tissue or organ in need of oxygen.
 2. Themethod of claim 1, wherein the protective border comprises polyethyleneglycol.
 3. The method of claim 1, wherein the lipid film is a monolayer.4. The method of claim 3, wherein the lipid envelope comprises at leastone base lipid and wherein the one or more emulsifying agents comprise aPEGylated lipid.
 5. The method of claim 1, wherein the oxygen isadministered via injection or as a continuous infusion.
 6. The method ofclaim 1, wherein the patient in need of treatment is experiencing localor systemic hypoxia.
 7. The method of claim 6, wherein the hypoxicconditions arise in the patient as a result of a disease or disorderselected from the group consisting of congenital physical or physiologicdiseases or disorders, embolisms, peripheral artery occlusive disease,transient ischemic attacks, strokes, acute trauma, surgicalinterventions, and exposure to chemical or environmental agents.
 8. Themethod of claim 1, wherein the suspension comprises 40 to 70% oxygen byvolume.
 9. The methods of claim 1, wherein the composition isadministered at a rate of 0.5 to 400 mL/minute.
 10. A method for forminga concentrated microbubble suspension comprising (a) forming amicrobubble suspension comprising a lipid envelope and a gas core,wherein the gas core comprises oxygen, wherein the lipid envelopecomprises one or more lipids in the form of a lipid film and one or moreemulsifying agents, wherein the outer surface of the lipid film forms aprotective border, wherein the one or more emulsifying agents comprisesa group or molecule that forms the protective border, (b) centrifugingthe microbubble suspension, and (c) separating the microbubbles based onsize to form a concentration microbubble suspension.
 11. The method ofclaim 10, wherein the concentrated microbubble suspension contains atleast four times the volume of oxygen that was present in themicrobubble suspension formed in step (a).
 12. The method of claim 10,wherein the microbubbles in the concentrated microbubble suspension havediameters of 20 microns or less.
 13. A composition for rapidlydelivering oxygen to a patient in need thereof comprising a suspensioncomprising microbubbles and a carrier, wherein the microbubbles comprisea lipid envelope and a gas core, wherein the gas core comprises oxygen,wherein the lipid envelope comprises one or more lipids in the form of alipid film and one or more emulsifying agents, wherein the outer surfaceof the lipid film forms a protective border, wherein the one or moreemulsifying agents comprises a group or molecule that forms theprotective border, and wherein the suspension comprises at least 40%oxygen by volume.
 14. The composition of claim 13, wherein theprotective border comprises polyethylene glycol.
 15. The composition ofclaim 13, wherein the lipid envelope comprises at least one base lipidand wherein the one or more emulsifying agents comprise a PEGylatedlipid.
 16. The composition of claim 13, wherein carrier is suitable forinjection.
 17. The composition of claim 15, wherein base lipid is aphospholipid comprising acyl chains of less than 24 carbons.
 18. A kitfor rapidly delivering oxygen to a patient in need thereof comprising(a) one or more lipids and one or more emulsifying agent for formingmicrobubbles, (b) a pharmaceutically acceptable carrier, and (c) asource of oxygen, wherein when (a), (b), and (c) are combined to form amicrobubble suspension, the microbubbles comprise a lipid envelope and agas core, wherein the gas core comprises oxygen, wherein the lipidenvelope comprises one or more lipids in the form of a lipid film andone or more emulsifying agents, wherein the outer surface of the lipidfilm forms a protective border, wherein the one or more emulsifyingagents comprises a group or molecule that forms the protective border,and wherein the suspension comprises at least 40% oxygen by volume. 19.The kit of claim 18 comprising instructions for forming the microbubblesand administering the microbubbles to a patient, tissue or organ in needthereof.
 20. The kit of claim 19 in a sterile packaging.
 21. A systemfor rapidly delivering oxygen to a patient in need thereof comprising(a) a means for generating a suspension comprising microbubbles and acarrier, wherein the microbubbles comprise a lipid envelope and a gascore, wherein the gas core comprises oxygen, wherein the lipid envelopecomprises one or more lipids in the form of a lipid film and one or moreemulsifying agents, wherein the outer surface of the lipid film forms aprotective border, wherein the one or more emulsifying agents comprisesa group or molecule that forms the protective border, and wherein thesuspension comprises at least 40% oxygen by volume, and (b) means foradministering microbubbles continuously or discontinuously to a patient,tissue or organ in need thereof.
 22. A method for delivering one or moregases to a patient, comprising administering to the patient acomposition, comprising a suspension comprising microbubbles and acarrier, wherein the microbubles comprise a lipid envelope and a gascore, wherein the gas core comprises at least one gas, wherein the gasis not a fluorinated gas, wherein the lipid envelope comprises one ormore lipids in the form of a lipid film, and one or more emulsifyingagents, wherein the outer surface of the lipid film forms a protectiveborder, wherein the one or more emulsifying agents comprises a group ormolecule that forms the protective border, and wherein the suspensioncomprises at least 40% of the one or more gases by volume, wherein thecomposition is administered in an effective amount to increase theconcentration of the one or more gases in the patient's blood, tissue ororgan.